Methods of generating libraries of nucleic acid sequences for detection via fluorescent in situ sequencing

Abstract

The present disclosure provides a number of targeted nucleic acid FISSEQ library construction methods. Targeted FISSEQ can exhibit several benefits, such as enhanced sensitivity and/or shorter assay time in the detection, identification, quantification, and/or determining the nucleotide sequence of the target species, relative to “random” or “whole-omic” detection via FISSEQ.

What is claimed is: 
     
       1. A method for enhancing a hybridization reaction in a cell, comprising:
 (a) providing said cell and a reaction mixture, said reaction mixture comprising (i) a target nucleic acid molecule, (ii) a probe having sequence complementarity with a target sequence of said target nucleic acid molecule, and (iii) a hybridization reaction enhancing agent comprising a polymer backbone, wherein said hybridization reaction enhancing agent enhances a rate of a hybridization reaction between said target nucleic acid molecule and said probe having sequence complementarity with said target sequence of said target molecule, wherein said hybridization reaction enhancing agent comprises a functional group that facilitates inactivation of said hybridization reaction enhancing agent, wherein said hybridization reaction enhancing agent comprises a modified polyethylene glycol (PEG) comprising an ionic PEG, a cleavable PEG or a hydrating; a polyacrylic acid; a polyvinylsulfonic acid; or an alginate; and wherein said cell is integrated with a hydrogel matrix; and 
 (b) subjecting said reaction mixture to conditions sufficient to conduct said hybridization reaction between said target nucleic acid molecule and said probe having sequence complementarity with said target sequence of said target nucleic acid molecule, wherein during said hybridization reaction, said hybridization reaction enhancing agent facilitates said hybridization reaction between said target nucleic acid molecule and said probe having sequence complementarity with said target sequence of said target molecule; and 
 (c) subjecting said functional group to conditions sufficient to inactivate said hybridization reaction enhancing agent. 
 
     
     
       2. The method of  claim 1 , further comprising inactivating said hybridization reaction enhancing agent. 
     
     
       3. The method of  claim 2 , wherein inactivating said hybridization reaction enhancing agent comprises inactivating said functional group by rendering an ionic group of said functional group to have a neutral charge, or by reducing hydration functionality of a hydrating group of said functional group. 
     
     
       4. The method of  claim 1 , further comprising, subsequent to (b), conducting an enzymatic reaction. 
     
     
       5. The method of  claim 4 , wherein said enzymatic reaction comprises reverse transcription, ligation, or nucleic acid polymerization. 
     
     
       6. The method of  claim 4 , wherein said hybridization reaction enhancing agent enhances a rate of said enzymatic reaction. 
     
     
       7. The method of  claim 1 , wherein said functional group is a hydrating group. 
     
     
       8. The method of  claim 7 , wherein said hybridization reaction enhancing agent comprises a cleavable linker between said polymer backbone and said hydrating group. 
     
     
       9. The method of  claim 1 , wherein said functional group is cleavable. 
     
     
       10. The method of  claim 9 , further comprising cleaving said functional group. 
     
     
       11. The method of  claim 10 , further comprising washing said functional group away from said target nucleic acid molecule. 
     
     
       12. The method of  claim 11 , further comprising conducting an enzymatic reaction subsequent to cleaving said functional group. 
     
     
       13. The method of  claim 11 , wherein said enzymatic reaction comprises reverse transcription, ligation, or nucleic acid polymerization. 
     
     
       14. The method of  claim 1 , further comprising, prior to (b), generating said hydrogel matrix in said cell. 
     
     
       15. The method of  claim 1 , wherein said reaction mixture further comprises a buffer. 
     
     
       16. The method of  claim 15 , wherein said buffer comprises a blocking agent, which blocking agent impedes non-specific binding of probes to off-target sequences of said target nucleic acid molecule. 
     
     
       17. The method of  claim 1 , wherein said polymer backbone is an ionic polymer backbone. 
     
     
       18. The method of  claim 17 , wherein said hybridization enhancing agent comprises a polyionic, polyelectrolyte, hydrophilic, or hydrating polymer.